Thiol metabolism in the parasitic nematode Haemonchus contortus

University of Technology, Sydney. Faculty of Science.Haemonchus contortus is an important parasitic nematode, both economically and pathologically. The emergence of widespread drug resistance requires new drug or vaccine targets to be identified. The requirement of aerobic organisms to control damage caused by reactive oxygen species and, the increased necessity of parasites to overcome the host immune response, has led to the investigation of antioxidant systems as potential targets. This work examines the thioredoxin antioxidant system in H. contortus, specifically the thioredoxin reductase and peroxiredoxin enzymes, to characterise their activity and determine if they are potential targets for parasite control. H. contortus contains two TrxRs, a cytoplasmic enzyme HcTrxRl with a selenocysteine in the active site, similar to the mammalian TrxR, and a mitochondrial enzyme HcTrxR2 with a nematode unique active site. HcTrxRl showed broad activity with thioredoxins from E. coli, sheep, and H. contortus while HcTrxR2 had high activity with only the mitochondrial H. contortus thioredoxin 1. Importantly, HcTrxRl was found to be more sensitive to the black tea inhibitor theaflavin than the selenocysteine containing mammalian TrxR, demonstrating the differences in the enzymes susceptibilities to inhibitors. To determine the function of the TrxR enzymes in nematodes, knockout (KO) strains of Caenorhabditis elegans were examined. TrxRl -/-KO worms were more sensitive to free radical attack and also to the anthelmintic ivermectin; while TrxR2 -/- KO eggs were highly sensitive to sodium hypochlorite. This demonstrates that inhibition of these enzymes would sensitise the nematodes to the host's immune attack. H. contortus contains two peroxiredoxins, the mitochondrial HcPrxl and the cytoplasmic HcPrx2. The activity of both peroxiredoxins was specific for the thioredoxin system; however, both peroxiredoxins were also able to be regenerated by the glutathione system when coupled to the nematode specific H. contortus thioredoxin 5. Both enzymes were stable to high concentrations of hydrogen peroxide which demonstrates different functions to their mammalian counterparts. A specific inhibitor of these peroxiredoxins was also identified which has minimal mammalian cytotoxicity. HcPrxl was found to be involved in drug resistance while HcPrx2 was found to be secreted and highly immunogenic. Analysis of homologous genes in C. elegans showed that both peroxiredoxin KO worms were sensitive to free radical attack; however, only the cytoplasmic CePrx2 KO C. elegans were sensitive to external oxidants. Overall, this work adds to the knowledge of H. contortus biology and identifies the enzymes of the thioredoxin system as potential drug or vaccine targets for parasite control

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3′-diindolylmethane. 3,3′-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3′-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3′-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3′-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3′-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings

Continuous heating of a giant X-ray flare on Algol

Giant flares can release large amounts of energy within a few days: X-ray emission alone can be up to ten percent of the star's bolometric luminosity. These flares exceed the luminosities of the largest solar flares by many orders of magnitude, which suggests that the underlying physical mechanisms supplying the energy are different from those on the Sun. Magnetic coupling between the components in a binary system or between a young star and an accretion disk has been proposed as a prerequisite for giant flares. Here we report X-ray observations of a giant flare on Algol B, a giant star in an eclipsing binary system. We observed a total X-ray eclipse of the flare, which demonstrates that the plasma was confined to Algol B, and reached a maximum height of 0.6 stellar radii above its surface. The flare occurred around the south pole of Algol B, and energy must have been released continously throughout its life. We conclude that a specific extrastellar environment is not required for the presence of a flare, and that the processes at work are therefore similar to those on the Sun.Comment: Nature, Sept. 2 199

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity

© 2015 Al Khamici et al. The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function

Experiences of youth justice: youth justice discourses and their multiple effects

Interventions within youth justice systems draw on a range of rationales and philosophies. Traditionally demarcated by a welfare/justice binary, the complex array of contemporary rationales meld different philosophies and practices, suggesting a mutability that gives this sphere a continued (re)productive and felt effect. While it may be increasingly difficult to ascertain which of these discourses is dominant in different jurisdictions in the UK, particular models of justice are perceived to be more prominent (Muncie, 2006). Traditionally it is assumed that Northern Ireland prioritises restoration, Wales prioritises rights, England priorities risk and Scotland welfare (McVie, 2011; Muncie, 2008, 2011). However, how these discourses are enacted in practice, how multiple and competing rationales circulate within them and most fundamentally how they are experienced by young people is less clear. This paper, based on research with young people who have experienced the full range of interventions in the youth justice system in Northern Ireland examines their narratives of ‘justice’. It considers how different discourses might influence the same intervention and how the deployment of multiple rationalities gives the experience of ‘justice’ its effect

WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear Β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector Β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.Peer reviewe

Mid-infrared supercontinuum generation using dispersion-engineered Ge11.5As24Se64.5 chalcogenide channel waveguide

We numerically investigate mid-infrared supercontinuum (SC) generation in dispersion-engineered, air-clad, Ge11.5As24Se64.5 chalcogenide-glass channel waveguides employing two different materials, Ge11.5As24S64.5 or MgF2 glass for their lower cladding. We study the effect of waveguide parameters on the bandwidth of the SC at the output of 1-cm-long waveguide. Our results show that output can vary over a wide range depending on its design and the pump wavelength employed. At the pump wavelength of 2 µm the SC never extended beyond 4.5 µm for any of our designs. However, supercontinuum could be extended to beyond 5 µm for a pump wavelength of 3.1 µm. A broadband SC spanning from 2 µm to 6 µm and extending over 1.5 octave could be generated with a moderate peak power of 500 W at a pump wavelength of 3.1 µm using an air-clad, all-chalcogenide, channel waveguide. We show that SC can be extended even further when MgF2 glass is used for the lower cladding of chalcogenide waveguide. Our numerical simulations produced SC spectra covering the wavelength range 1.8-7.7 µm (> two octaves) by using this geometry. Both ranges exceed the broadest SC bandwidths reported so far. Moreover, we realize it using 3.1 µm pump source and relatively low peak power pulses. By employing the same pump source, we show that SC spectra can cover a wavelength range of 1.8-11 µm (> 2.5 octaves) in a channel waveguide employing MgF2 glass for its lower cladding with a moderate peak power of 3000 W

Patient-centric trials for therapeutic development in precision oncology

An enhanced understanding of the molecular pathology of disease gained from genomic studies is facilitating the development of treatments that target discrete molecular subclasses of tumours. Considerable associated challenges include how to advance and implement targeted drug-development strategies. Precision medicine centres on delivering the most appropriate therapy to a patient on the basis of clinical and molecular features of their disease. The development of therapeutic agents that target molecular mechanisms is driving innovation in clinical-trial strategies. Although progress has been made, modifications to existing core paradigms in oncology drug development will be required to realize fully the promise of precision medicine

High genetic diversity at the extreme range edge: nucleotide variation at nuclear loci in Scots pine (Pinus sylvestris L.) in Scotland

Nucleotide polymorphism at 12 nuclear loci was studied in Scots pine populations across an environmental gradient in Scotland, to evaluate the impacts of demographic history and selection on genetic diversity. At eight loci, diversity patterns were compared between Scottish and continental European populations. At these loci, a similar level of diversity (θsil=~0.01) was found in Scottish vs mainland European populations, contrary to expectations for recent colonization, however, less rapid decay of linkage disequilibrium was observed in the former (ρ=0.0086±0.0009, ρ=0.0245±0.0022, respectively). Scottish populations also showed a deficit of rare nucleotide variants (multi-locus Tajima's D=0.316 vs D=−0.379) and differed significantly from mainland populations in allelic frequency and/or haplotype structure at several loci. Within Scotland, western populations showed slightly reduced nucleotide diversity (πtot=0.0068) compared with those from the south and east (0.0079 and 0.0083, respectively) and about three times higher recombination to diversity ratio (ρ/θ=0.71 vs 0.15 and 0.18, respectively). By comparison with results from coalescent simulations, the observed allelic frequency spectrum in the western populations was compatible with a relatively recent bottleneck (0.00175 × 4Ne generations) that reduced the population to about 2% of the present size. However, heterogeneity in the allelic frequency distribution among geographical regions in Scotland suggests that subsequent admixture of populations with different demographic histories may also have played a role